EP0550638A1

EP0550638A1 - Medium for culture of mammalian cells

Info

Publication number: EP0550638A1
Application number: EP19910918096
Authority: EP
Inventors: Luciano Ramos; Amy Anne Murnane; Melvin Susumu Oka
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 1990-09-25
Filing date: 1991-09-20
Publication date: 1993-07-14
Also published as: EP0550638A4; PT99048A; IE913345A1; NZ239900A; MX9101254A; ZA917561B; CA2091443A1; JPH06500918A; AU662491B2; PT99048B; WO1992005246A1; AU8734891A; TW240247B

Abstract

Milieux sans sérum de culture de cellules mammifères comprenant un milieu de base synthétique destiné à une culture cellulaire mammifère; environ 0,1 à environ 10 grammes par litre de levure hydrolysée; environ 0,1 à environ 5 grammes par litre de dextran ou d'albumine; environ 2 à environ 20 milligrammes par litre d'insuline; 0 à environ 100 milligrammes par litre d'un composé choisi dans le groupe constitué de transferrine, fructose ferrique, citrate ferreux et sulfate ferreux; ainsi qu'un composant d'acide gras constitué d'acide oléique, d'acide linoléique et d'acide linolénique dans un rapport d'environ 0,6: 1: 0,14 milligrammes d'acide gras par litre.Mammalian cell culture serum-free media comprising a synthetic base medium for mammalian cell culture; about 0.1 to about 10 grams per liter of hydrolyzed yeast; about 0.1 to about 5 grams per liter of dextran or albumin; about 2 to about 20 milligrams per liter of insulin; 0 to about 100 milligrams per liter of a compound selected from the group consisting of transferrin, ferric fructose, ferrous citrate and ferrous sulfate; as well as a fatty acid component consisting of oleic acid, linoleic acid and linolenic acid in a ratio of about 0.6: 1: 0.14 milligrams of fatty acid per liter.

Description

MEDIUM FOR CULTURE OF MAMMALIAN CELLS

Field of the Invention

The present invention relates to the field of cell culture media. More particularly the invention relates to the field of mammalian cell culture media. Background of the Invention

Beyond a basal nutrient mixture of salts, sugars, amino acids, and vitamins, cells .in vitro have also been found to require for proliferation a supplement of poorly defined biological fluids or extracts. Because of availability and ease of storage, the most commonly used supplement is serum. The use of serum in cell culture media, however, has several disadvantages. Serum is comparatively expensive. Since serum is not a defined component, different lots of serum may vary in the concentration of compounds present and thus result in unpredictable culture growth. Serum may also be contaminated with viruses or mycoplasms. The protein in serum may complicate the purification of cell products from the culture medium. In efforts to overcome the disadvantages of serum containing medium, researchers have attempted to provide serum-free media by substituting defined or better characterized components for serum. Unfortunately, the complexity of serum and the differing growth requirements of different types of cells has made it difficult to provide such media. For reviews on serum-free media for mammalian cell culture see Rizzino et al. (1979) "Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture" Nutrition Reviews 37: 369-378; Barnes and Sato (1980) "Serum-free Cell Culture: a Unifying Approach*¹, Cell .22.: 649-655; Barnes and Sato (1980) "Methods for Growth of Cultured Cells in Serum-Free Medium", Analyt. Biochem. 102: 255-270; and Bodeker et al. (1985) "A Screening Method To Develop Serum-Free Culture Media For Adherent Cell Lines", Develop. Biol. Standard. .6_0: 93-100.

U.S. Patent 4,786,599 issued November 22, 1988 to Chessebeuf and Padieu discloses a serum-free animal tissue culture medium containing a mixture of six fatty acids and albumin or dextran. The medium is particularly adapted for the primary culture of rat liver epithelial cells and possibly in the presence of hormones and/or growth factors, for obtaining cell lines, in particular myeloma and hybridoma cell lines.

Media for the serum-free culture of Chinese hamster ovary cells (CHO) have been reported. Gasser et al (1985) In Vitro Cellular & Developmental Biology 2.1: 588-592 discloses a serum-free medium for the culture of CHO cells. The serum- free medium is composed of a 1:1 mixture of Ham's F12 and modified Eagle's minimum essential media supplemented with transferrin, insulin, and selenium. Mendiaz et al. (1986) In Vitro Cellular & Developmental Biology .22.: 66-74 discloses a serum-free medium for the culture of CHO cells composed of a basal medium supplemented with insulin, and ferric sulfate or transferrin, selenium, trace elements, calcium chloride, glutamine, linoleic acid, non-essential amino acids, and insulin. Pietrzkowski et al (1988) Folia Histochemica et

Cytobiologica 2_6: 123-132 report a serum-free medium for the culture of chick embryo cells containing dextran. Pietrzkowski and Korohoda (1988) Folia Histochemica et Cytobiologica _\ 143-154 report a serum-free medium containing dextran for the culture of chick embryo fibroblasts. In these two publications, the dextran was added to the medium to enhance cell attachment and spreading. Ohmori (1988) Journal of Immunological Methods 112: 227-233 reports a serum-free medium which is able to support primary antibody responses by cultured murine lymphocytes. This medium is based on a basal medium supplemented with β- cyclodextrin, insulin, transferrin, albumin, low density lipoprotein, putrescine and alanine.

It is an object of the invention to provide serum- free media for the culture of mammalian cells. It is also object of the invention to provide serum-free media for the culture of mammalian cells transformed to produce recombinant products that increase product yield. It is yet another object of the invention to provide serum-free media for the culture of CHO cells. R-innmai-γ of the Invention The present invention provides media for the culture of mammalian cells. The invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description. Detailed Description of the Invention The media of the invention are useful for the culture of mammalian cells. The media of the invention have been found to be useful in the culture of Chinese hamster ovary (CHO) cells, and HAK cells, a baby hamster kidney cell line. The media of the invention have been found not suitable for the culture of myeloma cell lines.

Cells may be grown in batch and continuous culture with the serum-frae media of the invention. CHO cells grown in the media of the invention reach higher cell density and show increased recombinant product secretion when compared to CHO cells grown in a serum-containing medium.

The cell culture media of the invention are prepared by adding components to a basal medium designed for mammalian cell culture. The media are prepared in accordance with standard procedures for preparing cell culture media. Suitable basal media include standard mammalian cell culture media such as Ham's medium, Waymouth MB 752/1 medium. Eagle's medium, Williams E medium, 199 medium and derived edia of the types MEM and MEMα and any combinations of these media. Other standard media used for the culture of mammalian cells are also suitable for use in the invention. A preferred basal medium is the basal medium of Example 1. The preferred basal medium supports cell growth and significantly reduces the size of cell clumps in the media during cell culture.

A yeast hydrolysate such as Yeastolate is added to the basal medium in the amount of from about 0.1 to about 10.0 grams per liter, preferably in an amount of about 5 grams per liter.

Albumin or dextran is added to the basal medium, in an amount of from about 0.1 to about 5.0 grams per liter. Preferably either bovine serum albumin or dextran having a molecular weight of about 500,000 is added to the basal medium. Bovine serum albumin is preferably added in the amount of from about 0.1 to about 0.5 grams per liter. Dextran having a molecular weight of about 500,000 such as Dextran T500 is preferably added to the basal medium in the amount from about 0.1 to about 1.0 grams per liter. Insulin is added to the basal medium in the amount of from about 2.0 to about 20 milligrams per milliliter, preferably in the amount of about 10 milligrams per liter.

Transferrin or transferrin substitute is added to the basal medium in the amount of from about 0 to about 100.0 micrograms per milliliter. Transferrin may be substituted in the medium with ferric fructose (from about 1.0 to about 10.0 milligrams per liter) , ferric citrate (from about 1.0 to about 100.0 milligrams per liter), or ferrous sulfate (from about 5.0 micromoles to about 200.0 micromoles per liter). A mixture of the fatty acids oleic, linoleic and linolenic are added to the basal medium in the ratio of oleic 0.6: linoleic 1: linolenic 0.14 milligrams per liter of medium. In preferred embodiments of the invention, keeping this ratio of fatty acids, oleic acid is preferably added to the basal medium in the amount of from about 0.012 to about 0.12 milligrams per liter; linoleic acid is preferably added to the basal medium in the amount of from about 0.2 to about 5.0 milligrams per liter; linolenic acid is added to the medium in the amount of from about 0.028 to about 0.7 milligrams per liter. Cholesterol is added to the basal medium in the amount of from about 0 to about 10.0 milligrams per liter.

In a preferred embodiment of the invention which is described in further detail in Example 2, calcium chloride

(CaCl₂) (anhydrous) is added to the basal medium in the amount of from about 0 to about 200 milligrams per liter, preferably in the amount of about 66.67 milligrams per liter. Magnesium sulfate (MgSO (anhydrous) is added to the basal medium in the amount of from about 0 to about 100.0 milligrams per liter, preferably in the amount of about 24 milligrams per liter.

The pH of the medium is preferably from about 6.8 to about 7.4. The osmolarity of the medium is preferably from about 280 to 360 milliosmoles.

The basal medium may be stored as a powder at 4^*C for one year. The complete medium (basal medium with added supplements) in a liquid form may be stored at 4^βC for six months.

Preferred embodiments of the invention are described in the following Examples. Example 1 Preparation of Basal Medium

The components in the basal media are mixed and ball-mill ground to formulate a homogeneous powder. The powdered media is then dispensed into 100L packets and stored at 4'C.

BASAL MEDIUM COMPONENTS; MR1 SERUM-FREE MEDIA COMPONENTS milligrams/1iter

INORGANIC SALTS/TRACE ELEMENTS

NaCl

KCL

NaH2PO4.H20

Na2HP04

MgC12 6H20

MgS04 (anhydrous)

CUSO4.5H20

Fe(NO3)3.9H20

FeSO4.7H20

ZnSO4.7H20

MnC12.4H20

Na2Se03 (anhyd)

AMINO ACIDS

L-Alanine 41. 300000 L-Arginine HCl 112.546700 L-Arginine FB 16.666000 L-Asparagine H20 28.336700 L-Aspartic Acid 24.433300 L-Cystine 2HC1 19.116600 L-Cysteine HC1.H20 45.040000 L-Cysteine FB 13.333300 L-Glutamic Acid 46.566700 L-Glutamine 292.000000 Glycine 35.833300

L-Histidine HC1.H20 20.986700 L-Histidine FB 5.000000 L-Isoleucine 35.480000 L-Leucine 46.833300 L-Lysine HCl 65.486600 L-Methionine 11.493300 L-Phenylalanine 20.653300 L-Proline 34.833300 L-Serine 15.166700 L-Threonine 33.300000 L-Tryptophan 7.346700 L-Tyrosine 2Na2H20 36.776700 L-Valine 35.900000

VITAMINS/MISC. COMPONENTS

Dextrose 4500.000000

Putrescine 2HC1 0.053700

Sodium Pyruvate 81.666700

Ascorbic Acid 17.333300

Biotin 0.202400

D-Calcium Pantothenate • 0.160000

Sodium Pantothenate 0.337330 -7-

Choline Chloride 5.486700

Folic Acid 1.100000 i-Inositol 7.333300

Nicotinamide 0.679000 Na2 alpha Tocopherol P04 0.003300

Glutathione (Reduced) 0.016700

Menadione Na Bisulfite 0.003300

Pyridoxine HCl 0.020700

Pyridoxal HCl 0.666700 Riboflavin 0.079300

Thiamine HCl 0.780000

Vitamin B12 0.973300

Calciferol 0.033300

Methyl Linoleate 0.010000 Vitamin A Acetate 0.033000

Linoleic Acid 0.028000

Lipoic Acid 0.136700

Preparation of Basal Medium - for a final volume of 100L Ninety liters of deionized-distilled water is measured into an appropriate mixing vessel. One 100L packet of ball-mill ground powdered media (see above) is added. The pH of the medium is adjusted to 7.2 using IN HCl. The volume of the medium is brought to 100L by the addition of water. The medium may then be sterilized by membrane filtration using a 0.2 micron cellulose acetate filter. Example 2 Preparation of Medium MRl-3

Medium MRl-3 contains the basal medium of Example 1 supplemented with 5,000 mg/1 TC Yeastolate (Difco, Detroit, Michigan) , 500 mg/1 bovine serum albumin (BSA) (Armour, Kankakee, Illindis) 10 mg/1 bovine insulin (Waitaki, Toronto, Canada), 10 mg/1 bovine transferrin (Sigma Chemical Co., St. Louis, Missouri), 0.12 mg/1 oleic acid (Ameresco, Cleveland, Ohio), 0.20 mg/1 linoleic acid (Ameresco), 0.028 mg/1 linolenic acid (Ameresco) , 2 mg/1 cholesterol (Ameresco) , 66.67 mg/1 anhydrous calcium chloride, and 24 mg/1 anhydrous magnesium sulfate. The medium is prepared as follows:

For a final volume of 100L

1. Measure 90 liters of deionized-distilled water into an appropriate mixing vessel. 2. Add one 100L packet of ball-mill ground powdered media (from Example 1) .

3. Add 2.4 grams of MgS0₄ (anhydrous) and mix until dissolved.

4. Add 6.7 grams of CaCl₂ (anhydrous) and mix until dissolved.

5. Add 500 grams of TC Yeastolate, mix until dissolved.

6. Add 50 grams of BSA, mix until dissolved.

7. Add 220 grams of NaHC0₃, mix until dissolved. 8. Add 1 gram of insulin, 1 gram of transferrin (or 100 ml of ferric fructose) and mix until dissolved.

9. Dissolve 12 mg of Oleic acid, 20 mg of Linoleic acid, 2.8 mg of Linolenic acid, and 200 mg of cholesterol in 100 mis of absolute ethanol, and add this fatty acid mix to the mixing vessel.

10. Adjust the pH to 7.2 using IN HCl.

11. Bring the volume to 100 liters and mix thoroughly.

12. Filter sterilize using a 0.2 micron cellulose acetate filter. 13. Check osmolarity and record.

14. Store at 4^βC for up to six months.

Example 3 Preparation of Medium MR1-6

Medium MR1-6 is contains the basal medium of Example 1 supplemented with 5,000 mg/1 TC Yeastolate (Difco, Detroit, Michigan) , 500 mg/1 bovine serum albumin (Armour, Kankakee, Illinois) , 10 mg/1 bovine insulin (Waitaki, Toronto, Canada) , 10 mg/1 bovine transferrin (Sigma Chemical Co., St. Louis, Missouri), 0.12 mg/1 oleic acid (Ameresco, Cleveland, Ohio), 0.20 mg/1 linoleic acid (Ameresco), 0.028 mg/1 linolenic acid (Ameresco) , and 2 mg/1 cholesterol (Ameresco) . The medium is prepared in the same way as medium MRl-3 in Example 2 except that steps 3 and 4 are omitted. In this medium no additional MgS0₄ or CaCl₂ is added. Example 4 Preparation of Medium MRl-7. Medium MRl-7 contains the basal medium of Example 1 supplemented with 5,000 mg/1 TC Yeastolate (Difco, Detroit, Michigan), 1,000 mg/1 Dextran T-500 (Pharmacia, Piscataway, New Jersey), 10 mg/1 bovine insulin (Waitaki, Toronto, Canada) , 10 mg/1 bovine transferrin (Sigma Chemical Co, St. Louis, Missouri) , 0.12 mg/1 oleic acid (Ameresco, Cleveland, Ohio), 0.20 mg/1 linoleic acid (Ameresco), 0.028 mg/1 linolenic acid (Ameresco) , and 2 mg/1 cholesterol (Ameresco) . Mediun MRl-7 is prepared in the same way as medium MRl-3 in Example 2 except that steps 3 and 4 are omitted and Dextran T-500 replaces bovine serum albumin in step 6. At step 6, 100 grams of Dextran T-500 are added and mixed until dissolved. ^•By.»ιtι ^*ι«» 5 Cell Culture

CHO cells transformed to produce soluble T4, a soluble form of the T-4 lymphocytic cell receptor (cell line 37-80N) , were cultured in four different media: serum containing medium Alpha (-) MEM/5% Fetal bovine serum (FBS) , and the media described in Examples 2, 3, and 4. 5 x 10⁵ cells per milliliter were cultured for 7 days after seeding in 250 ml SP flasks with 150 ml of medium. Total cell number was determined by Coulter counter, and viability was determined by trypan blue dye exclusion using a hemocytometer. Concentration of ST4 was determined by an ELISA-based assay. At day two after seeding, the serum-free media showed greater number of cells than the serum containing medium. In serum- containing medium, there were approximately 1.3 x 10⁶ cells, whereas in the serum-free media there were approximately 1.6 x 10⁶ cells. At days 3 through 7 significantly more cells were present in the serum-free media than the serum containing medium. At day 3, there were approximately 2.4 x 10⁶ cells in the serum-containing medium and approximately 3.3 x 10⁶ cells in the serum-free media. At day 4, the total number of cells in the serum-containing medium had dropped slightly to about 2.25 x 10⁶ cells. In contrast, the number of cells in the serum-free media had increased to approximately 3.6 x 10⁶ cells in MRl-7, 4.1 x 10⁶ cells in MRl-3, and 4.3 x 10⁶ cells in MR1-6. By day 7, the total number of cells in medium MR1- 7 had increased to approximately 4.0 x 10⁶ cell, and the number of eels in the other media remained at levels comparable to the levels at day 4.

By three days post seeding, cells grown in the seru - free media produced significantly more sT4 than did cells grown in the serum containing medium. The difference in amount of sT4 product became more pronounced at days 4-7. At day 7, cells cultured in the serum free media produced from about 75 to 87 micrograms of sT4 per milliliter of medium, whereas cells cultured in the serum containing medium produced about 35 micrograms of sT4 per milliliter of medium.

Claims

1. A serum-free mammalian cell culture medium comprising:

(a) a synthetic basal medium designed for mammalian cell culture;

(b) about 0.1 to about 10 grams per liter hydrolyzed yeast;

(c) about 0.1 to about 5 grams per liter of dextran or albumin;

(d) about 2 to about 20 milligrams per liter insulin;

. (e) 0 to about 100 milligrams per liter of a compound selected from the group consisting of transferrin, ferric fructose, ferrous citrate and ferrous sulfate; and

(f) a fatty acid component consisting of oleic acid, linoleic acid and linolenic acid in a ratio of about 0.6 : 1 : 0.14 milligrams of fatty acid per liter.

2. The serum free mammalian cell culture medium of claim 1 further comprising 0 to about 10 milligrams per liter cholesterol.

3. The serum-free mammalian cell culture medium of claim 1 further comprising 0 to about 200 milligrams per liter anhydrous calcium chloride and 0 to about 100 milligrams per liter anhydrous magnesium sulfate.

4. The medium of claim 1 wherein said hydrolyzed yeast is present in the medium in the amount of about five grams per liter.

5. The medium of claim 1 wherein albumin is present in said medium in the amount of about 0.5 grams per liter.

6. The medium of claim 5 wherein said albumin is bovine serum albumin.

7. The medium of claim 1 wherein said dextran is present in said medium in the amount of about one gram per liter.

8. The medium of claim 7 wherein said dextran is dextran having a molecular weight of about 500,000.

9. The medium of claim 1 wherein said insulin is present in said medium in the amount of about 10 milligrams per liter.

10. The medium of claim 1 wherein transferrin is present in -li¬ the amount of about 10 milligrams per liter.

11. The medium of claim 1 wherein oleic acid is present in the amount of about 0.12 milligrams per liter; linoleic acid is present in the amount of about 0.20 milligrams per liter; and linolenic acid is present in the amount of about 0.028 milligrams per liter.

12. The medium of claim 2 wherein cholesterol is present in the amount of about two milligrams per liter.

13. The medium of claim 3 wherein said calcium chloride is present in the amount of about 66 to about 67 milligrams per liter; and magnesium sulfate is present in the amount of about 24 milligrams per liter.

14. A serum-free mammalian cell culture medium comprising:

(a) a synthetic basal medium designed for mammalian cell culture;

(b) about 5 grams per liter hydrolyzed yeast; (c) about 1 gram per liter of albumin;

(d) about 10 milligrams per liter insulin;

(e) about 10 milligrams per milliliter transferrin;

(f) a fatty acid component consisting of about 0.12 milligrams per liter oleic acid, about 0.20 milligrams per liter linoleic acid and about 0.028 milligrams per liter linolenic acid; and

(g) about 2 milligrams per liter cholesterol;

15. The medium of claim 14 further comprising about 66 to about 67 milligrams per liter anhydrous calcium chloride; and about 24 milligrams per liter anhydrous magnesium sulfate.

16. A serum-free mammalian cell culture medium comprising:

(a) a synthetic basal medium designed for mammalian cell culture;

(b) about 5 grams per liter hydrolyzed yeast; (c) about 1 gram per liter dextran having a molecular weight of about 500,000;

(d) about 10 milligrams per liter insulin;

(e) about 10 milligrams per liter transferrin;

(g) about 2 milligrams per liter cholesterol.